Isolation and characterisation of rainbow trout, Oncorhynchus mykiss, leucocyte subpopulations using monoclonal antibodies
Attempts were made to generate monoclonal antibodies (MoAb) against rainbow trout, Oncorhynchus mykiss macrophages, using macrophage membrane preparations for immunisation. Although no successful cloning was achieved, positive hybridomas were generated and appeared to secrete antibodies to leucocyte subpopulations. Further studies utilised the MoAb E3D9, I-14, C4B6 and G2F6 to examine the effects of stress and challenge (with virulent and avirulent strains of Aeromonas salmonicida) on the subpopulations of peripheral blood leucocytes (PBL) recognised by these MoAb. On the basis of morphological and functional studies it was determined that E3D9 is a neutrophil marker, C4B6 recognises a subpopulation of lymphocytes and granulocytes and G2F6 antibody recognises a large proportion of granulocytes and a small but significant proportion of lymphocytes. The I-14 antibody is a surface Ig marker (DeLuca et al., 1983). The E3D9+ cells increased in all three trails to twice the pre-trial levels in the total bitmap by the end of the trials and during the challenge trial the antigen(s) recognised by the antibody E3D9 was downregulated whereas in the stress trial the antigen was upregulated. In the case of C4B6, by the end of the stress trial numbers of C4B6+ cells were half the pre-stress level and the fluorescent intensity during the Brivax challenge was greater at the end of the trial than the pre-challenge level in contrast to the virulent challenge where the reverse was true. Also, the antigen recognised seemed to be present in large numbers on the surface of the C4B6+ cells which is in contrast to the G2F6 antibody where there seemed to be few antigens present on the surface. There were no significant population changes during the trials with the G2F6+ cells in the total bitmap, however, in the granulocyte bitmap during the challenge trial using the virulent strain there was a large decrease in the percentage of G2F6+ cells throughout the trial and it was only on the last day of the trial that they started increasing again.